Pharmacological Studies on Novel Triazino Quinolines

Pharmacological importance of the nitrogen heterocycles is countless. The triazines are found to possess exceptional biological antitumor, anti-HIV, antiviral, antimalarial, antimicrobial, cytotoxic activities. The current investigation attempts to evaluate invitro antibacterial, antifungal, antioxidant potential, and cytotoxicity of newly synthesized substituted 4'-methyl-3-thioxo-1,2,4-triazinoquinolin-5-one. The antimicrobial activity was done by Agar Well Diffusion Method and the MIC of the compound was found using the Broth dilution assay method. The compounds showed excellent antibacterial activity against selected bacterial strains, including Gram-positive S. aureus, S. pyogens , and Gram-negative bacteria P. aeroginosa, E. coli, K. peumoniae, Pseudomonas Sp with the zones of inhibition 9 to 19nm. The standard drug Ampicillin showed a maximum inhibitory zone 18 nm. Among all the screened compounds, sample exhibited good activity. Similarly, the compounds were screened for antifungal properties, which showed an excellent reduction in the growth of selected fungal strain for Candida albicans. The compounds were also screened for 1,1-diphenyl-2-picrylhydrazyl (DPPH) activity. Cytotoxicity was done in Dalton’s Lymphoma Ascites (DLA) cells which were obtained from Amala Cancer Research Center. The tested compounds exhibited significant antioxidant activity in a concentration-dependent manner.


Introduction
The compounds with quinoline moiety are well branded due to their extensive biological activity [1]. In particular, 8-hydroxyquinoline and its derivatives were established into antifungal clinical use and novel compounds of this type are still investigated [2][3][4]. The utility of quinoline derivatives in the areas of medicine [5] is well established. These compounds are found to possess various bioactivities such as anti-malarial [6-8], anti-bacterial [9][10][11][12], anti-fungal [13][14][15], and anti-cancer agents [16][17][18][19]. The literature survey revealed that synthetic quinoline and substituted quinoline derivatives are associated with a wide range of biological properties in curing a lot of diseases.
Over the last three decades, there has been a dramatic increase in the incidence of fungal infections. The Discovery of new drugs for the treatment of systemic mycoses is a major challenge in infectious disease research. There is an urgent need for new antifungal remedies with novel modes of action due to a decreased antifungal susceptibility of newly emerging fungi in the growing set of immune compromised patients [20][21][22].
It is well known that most of the heterocyclic compounds which have a compact structure possess antioxidant, properties [23][24][25].

Drugs
The compounds tested were prepared in our laboratory and their structures were characterized by FTIR, 1 H-NMR and 13 C-NMR spectral techniques. Antimicrobial activity

Agar Well Diffusion Method
The agar well diffusion method [42] is used to determine growth inhibition.
The results were observed and recorded. It was compared with the standard antimicrobial agent ampicillin.

Minimum Inhibitory Concentration (MIC)
The minimum inhibitory concentration of the compounds was found using the broth dilution assay method (NCCLS, 1997). After incubation result was observed and tabulated.
Seven test tubes containing 1mL of sterile nutrient broth for bacterial species and Sabouraud's dextrose broth for yeast and fungal species were prepared.
For assaying the drugs, the starting concentration is kept at 8 mg/mL in the first tubes containing 1mL of broth.
Tubes were vortex mixed thoroughly to make the initial standard concentration. This was serially diluted to other tubes and finally, 1mL is discarded from the seventh test tube. So, the dilution will be 8, 4, 2, 1, 0.5, 0.25, 0.125 mg/mL respectively. To all these test tubes, 0.1mL of the cultures of the target microorganism was added separately and incubated at 37˚C for 24-48 hours. After incubation, a loop full of samples from the test tubes was inoculated in the selective medium for the bacterial species and Sabouraud's Dextrose Agar for yeast or fungal species and incubated for 24 hours at 37˚C for 24 hours for bacteria and room temperature for yeast for 24 hours.
After incubation result was observed and recorded.

Antioxidant activity
2, 2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay The DPPH radical scavenging assay was determined according to Leong and Shui (2002). Methanol was used as both a blank and negative control. Results were observed and tabulated.

Cell Line
Dalton's Lymphoma Ascites (DLA) cells were obtained from Amala Cancer Research Center. The cells were maintained by transplanting every 2 weeks as Ascites in the peritoneal cavity of Swiss albino mice. The test compounds were studied for in vitro cytotoxicity using Dalton's lymphoma ascites cells (DLA).
The tumor cells aspirated from the peritoneal cavity of tumor-bearing mice were washed thrice with PBS or normal saline. Cell viability was determined by the trypan blue exclusion method. Viable cell suspension (1x106 cells in 0.1mL) was added to tubes containing various concentrations of the test compounds and the volume was made up to 1mL using phosphate-buffered saline (PBS). The Control tube contained only cell suspension. These assay mixtures were incubated for 3 hours at 37 0 C. The further cell suspension was mixed with 0.1mL of 1% trypan blue and kept for 2-3 minutes and loaded on a hemocytometer. Dead cells take up the blue color of trypan blue while live cells do not take up the dye the chamber of stained and unstained color is counted separately.

Results and Discussion
Antibacterial activity: To check the biological activity of the compounds, the series of the various substituted triazino quinolines were screened for antimicrobial activity against gram-positive bacteria P. aeroginosa, E.coli, K. peumoniae gram negative bacteria S. aureus, and S. Pyogens and antifungal activity against Candida Albicans in DMSO by well diffusion method. The activity of all the synthesized compounds was calculated against the corresponding standard drug. The products showed various activities against all species of microorganisms which suggest that the variations in the structures affect the growth of the microorganisms. From Table 1 it is evident that all the tested compounds showed potential antibacterial activity against all the bacteria employed and 7'-chloro-4'-methyl-3-thioxo 1,2,4-triazino-quinolin-5-one 1f is more active towards S. aureus, P. auroginosa as well as S. Pyogen, 8'-chloro-4'-methyl-3-thioxo 1, 2, 4-triazino-quinolin-5-one 1c against E.coli acted as a good inhibitor at 1µg/mL. This may be because of the presence of chlorine atoms present in the ring. Thus, it can be concluded from these results that the prepared compounds showed a moderate to high antimicrobial activity towards all species of bacteria. Antifungal studies were also done for the synthesized compounds against Candida albicans and tabulated in Table 2. The screening data of antifungal activity of these series of compounds show moderate to good antifungal activity. It infers that 4'-methyl-3-thioxo 1, 2, 4-triazino-quinolin-5-one 1e performed maximum inhibitory action against the fungi Candida albicans and the compound '-chloro-4'-methyl-3-thioxo 1, 2, 4-triazino-quinolin-5-one 1c exhibits no inhibition at lower concentration compared to the other compounds.

Cytotoxicity results:
The cytotoxicity measurement is one of the most important indicators of in vitro biological evaluation system. Cytotoxicity of the selected triazino quinolines was studied in vitro using Dalton's Lymphoma Ascites (DLA) cells [4][5][6][7] . The compounds were tested at five different concentrations. After 24 hours the results obtained were compared with that of control, percentage cell death and IC 50 were calculated. Table 5 shows the percentage cell death for substituted 4'-methyl-3-thioxo-1,2,4-triazinoquinolin-5-one (1a-f). From the table, it is the LC50 values was found to be 35,34,67,18,30,46 µg /mL, respectively. It is found that the value is maximum for 4'-methyl-3-thioxo-1,2,4-triazinoquinolin-5-one (1e). It was noticed that when we increased the drug concentration the percentage of cell death also increased.

Conclusion
The pharmacological studies such as invitro antibacterial, antifungal and, antioxidant potential, and cytotoxicity of newly synthesized triazino quinolines were carried out. Antibacterial studies were done against various gram-positive and gram-negative bacteria stains. S. Pyogens, S. aureus is the gram-positive bacterias, and E.coli, P. aeruginosa and, K. peumoniae are the gram-negative bacterias used. Cytotoxicity was done in Dalton's Lymphoma Ascites (DLA) cells were obtained from Amala Cancer Research Center. Antioxidant activity was done in 2, 2-Diphenyl-1-picrylhydrazyl radical scavenging assay. Antimicrobial activity was done by Agar Well Diffusion Method and the MIC of the compound was found using the Broth dilution assay method. The tested compounds were found to have good antibacterial antifungal and antioxidant activities. Cytotoxicity of tested compounds was found to have potential activity. From the above results, we can conclude that triazino quinoline has potential health benefits. [11] Chevalier J., Atifi S., Eyraud A., Mahamoud A., Barbe J., Pages J.M., New pyridoquinoline derivatives as potential